The Mysterious Multi-Modal Repellency of DEET

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2015 The ysm terious multi-modal repellency of DEET Matthew eGeD nnaro Biomolecular Sciences Institute, Florida International University, [email protected]

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Recommended Citation DeGennaro, M. (2015). The ysm terious multi-modal repellency of DEET. Fly, 9:1, 45-51

This work is brought to you for free and open access by the College of Arts, Sciences & Education at FIU Digital Commons. It has been accepted for inclusion in Biomolecular Sciences Institute: Faculty Publications by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. REVIEW Fly 9:1, 45--51; January/February/March 2015; Published with license by Taylor & Francis Group, LLC The mysterious multi-modal repellency of DEET

Matthew DeGennaro*

Biomolecular Sciences Institute & Department of Biological Sciences; Florida International University; Miami, FL USA

Keywords: chemosensation, confusant, DEET, kairomone, Ir40a, mosquito, olfaction, Orco, receptors, repellent

5,6 DEET is the most effective insect repellent available and insecticides, miticides, and repellents. Potential repellents has been widely used for more than half a century. Here, I against Aedes aegypti were identiﬁed by testing a diverse set of review what is known about the olfactory and contact 6,241 compounds. Aedes aegypti, the vector for yellow and den- mechanisms of DEET repellency. For mosquitoes, DEET has gue fever as well as chikungunya, was selected because its behav- at least two molecular targets: Odorant Receptors (ORs) ior is easier to assay in the laboratory than that of other disease mediate the effect of DEET at a distance, while unknown transmitting mosquitoes. During screening, 1 ml of each test chemoreceptors mediate repellency upon contact. compound was distributed on the forearms, and the hands were Additionally, the ionotropic receptor Ir40a has recently been placed in cages containing 2,000–4,000 mosquitoes. Based on ﬁ Drosophila identi ed as a putative DEET chemosensor in . The the protection time, 56% of 4,137 tested compounds were effec- mechanism of how DEET manipulates these molecular tive for less than 1 h, 28% for 1–2 h, 7% for 2–3 h, and 9% for targets to induce insect avoidance in the vapor phase is also more than 3 h. In separate experiments, cloths were impregnated contested. Two hypotheses are the most likely: DEET 2 activates an innate olfactory neural circuit leading to with 3.6 mg / cm of one of 3,239 compounds, and then placed avoidance of hosts (smell and avoid hypothesis) or DEET has on the forearms. 51% of the compounds were effective for less no behavioral effect on its own, but instead acts than 1 d, 16% for 1–5 d, 8% for 5–10 d, and 25% for more cooperatively with host odors to drive repellency (confusant than 10 d. N,N-diethylbenzamide was among the most effective hypothesis). Resolving this mystery will inform the search for compounds found, repelling for more than 3 h when applied to a new generation of insect repellents. skin and 10 d when applied on cloth. However, it also caused 7

skin irritation. Determined to find a repellent that was not an irritant, 7 DEET is the most effective invertebrate repellent to prevent 33 derivatives from N,N-diethylbenzamide were created. All mosquitoes, flies, ticks, and even parasitic worms from feeding toluic acid derivatives, including N,N- diethyl-3-methylbenza- on humans.1-4 How a single chemical can change the normal mide (i.e., N,N-diethyl-m-toluamide) repelled mosquitoes when 7,8 behavioral response to otherwise attractive stimuli is of consider- applied on skin or cloths. LD 50 of N,N-diethyl-m-toluamide able interest to neuroscientist, or anyone who seeks to prevent in rats was very low (2 g/kg), with no evidence of systemic toxic- 9 the transmission of vector-borne disease. DEET is not without ity upon frequent dermal application or inhalation. Subsequent its drawbacks, and understanding the molecular mechanism of studies showed that N,N-diethyl-m-toluamide is safe for human 10 DEET’s action has great potential for the development of more use, but it was recommended that ingestion be avoided. N,N- effective repellents. Recent studies have suggested multiple modes diethyl-m-toluamide was renamed DEET by the Committee on of action for DEET repellency. Given the controversy in the Insecticide Terminology of the Entomological Society of Amer- field, I seek to provide a context to published results and suggest ica, because of “numerous complaints that diethyltoluamide was 11 directions for future research. too long for a common name.” DEET was registered in the United States for use by the general public in 1957, and reregis- tered in 1998 (US EPA document EPA 738-R-98–010). There The Discovery of DEET are approximately 120 products currently on the market that contain DEET at concentrations from 4% to 100%. In 1942, the United States Department of Agriculture (USDA), in collaboration with the US military, screened more than 7,000 compounds over a 5-year period to develop Theories of Repellency

© Matthew DeGennaro Early studies suggested that repellents target the central ner- *Correspondence to: Matthew DeGennaro; Email: mdegenna@fiu.edu Submitted: 04/28/2015; Revised: 07/03/2015; Accepted: 07/20/2015 vous system (CNS), the peripheral nervous system (PNS), or http://dx.doi.org/10.1080/19336934.2015.1079360 both. Five theoretical modes of action for insect repellents were This is an Open Access article distributed under the terms of the Creative proposed: 1) inhibiting the response of sensory neurons of host Commons Attribution-Non-Commercial License (http://creativecommons. attractants, 2) activating a receptor system that mediates a com- org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, peting or inappropriate behavior, 3) acting as attractant at low distribution, and reproduction in any medium, provided the original work is concentration, but as a repellant at high concentration 4) activat- properly cited. The moral rights of the named author(s) have been asserted. ing receptors linked to several behavioral programs to increase www.tandfonline.com Fly 45 the noise/signal ratio in order to “jam” the relevant sensory cir- DEET requires insect ORs to repel in the vapor phase cuit, and 5) activating unique avoidance/aversive receptor(s).12 From the 1970s onward, it was clear that DEET changed the These hypotheses informed later studies but were conceived olfactory responses of insects, but the molecular mechanism was before olfactory receptors were identified. They provided a con- unknown. Beginning in 1999, insect olfactory receptors were ceptual framework for the current proposed molecular mecha- identified and the search for the molecular target(s) of DEET nisms of DEET repellency. began.25-30 Insect olfactory sensilla usually contain 2 or more An additional model for DEET repellency was based purely olfactory receptor neurons that respond to distinct odors due to on its molecular structure. It suggested that DEET, which is non- the different olfactory receptors they express. We now know that polar, interacts with lipids of the cell membrane of chemosensory insects use at least 3 families of olfactory receptors to smell: odor- neurons.13 Here, DEET is proposed to have an indirect effect on ant receptors (ORs), ionotropic receptors (IRs), and gustatory olfactory receptor activation by altering membrane excitability or receptors (GRs).31,32 A small number of GRs have been identi- function. If the cell membrane is altered by DEET, the change fied that respond to carbon dioxide,20,28,33 but surprisingly, a must be extremely transient, as electrophysiological studies of few GRs known to detect sweet compounds in taste neurons insect sensilla have shown rapid recovery of olfactory receptor were also found to be expressed in select olfactory receptor neu- neurons to baseline spike activity after the application of rons.34 IRs and ORs25,26,29 respond to a broad spectrum of odor- DEET.14-16 Studies uncovering DEET-insensitive mutants fur- ants. For example, in Aedes aegypti there are 131 odor-selective ther challenged this model by showing that DEET had particular ORs.30 In Drosophila melanogaster and Aedes aegypti, DEET molecular targets and did not have a promiscuous effect on cell repellency has been clearly shown to require Orco,15,35 the obli- membrane function.15,17,18 However, this lipid interaction gate co-receptor for the OR family of odor-gated ion channels.36- model was prescient in suggesting that DEET could modulate 39 In addition, natural variation in Drosophila Or59B was shown the activity of multiple olfactory receptors and thereby disrupt to change the receptor’s electrophysiological response to 1-octen- the odor coding necessary for host detection. 3-ol when co-presented with DEET. Loss of receptor sensitivity to DEET was mapped to a change in just one amino acid (valine 91 to alanine).18 These genetic studies present strong evidence Olfactory Mechanism of Action that both ORs and Orco are required for insects to sense DEET, but they do not reveal which of the OR(s) are the behaviorally Although the volatility of DEET is not particularly high relevant molecular DEET targets. (0.00167 mmHg at 25C) compared to many host odors such as lactic acid (0.0813 mmHg at 25C) or 1-octen-3-ol (0.53 ORs and behavioral inhibition mmHg at 25C), it is effective in the vapor phase. Laboratory ORs are molecular targets of DEET, but how DEET interacts studies have focused on identifying the molecular target(s) of with ORs to change insect behavior is an area of active investiga- tion. The initial genetic analysis of Drosophila behavior suggested DEET using a combination of behavioral genetics and electro- 15 physiological approaches. These studies have led to 3 main that DEET inhibited odor detection via the OR pathway. Loss hypotheses to explain the mode of action of DEET: the inhibi- of Orco allowed flies to enter food-baited traps that are perfumed tion of host odor detection hypothesis, the confusant hypothesis, with 10% DEET that wild-type flies avoid. If food was absent and the smell and avoid hypothesis (Fig. 1). from the traps, wild-type flies entered DEET perfumed traps. This result suggests that the presence of food odors is required for DEET’s ability to repel flies. However, DEET could repel Inhibition of lactic acid sensation without food odors at high concentrations, particularly when flies It is possible that a repellent like DEET could work by mask- were able to contact DEET. Electrophysiological studies in mos- ing a host odor, thereby decreasing the ability of the insect to quito and fly sensilla and experiments with heterologously detect its feeding target. Lactic acid is a human odor that can expressed receptors showed that DEET can inhibit the responses attract mosquitoes particularly when co-presented with other of a subset of ORs to their odor-ligands.15 This inhibitory effect kairomones, such as carbon dioxide.19,20 Electrophysiological extended to non-selective cation channels, such as Drosophila studies showed that DEET reduces the sensitivity of olfactory Ether-a-go-go and mouse TRPM8. However, not all ORs or ion receptor neurons to odors by decreasing the responses of lactic channels tested were inhibited by DEET, suggesting that DEET acid-excited neurons and increasing the inhibition of lactic acid- possesses some selectivity. Combining their electrophysiological inhibited neurons.21 Behavioral assays in a repellometer con- and behavioral data, Ditzen et al.15 concluded that DEET acts in firmed that DEET inhibited the attraction of Aedes aegypti to lac- the vapor phase to inhibit the detection of attractive odors. How- tic acid, but both compounds were also attractive to the ever, the observations made in these studies have since been mosquito.22 Thus, the authors of these studies classified DEET reinterpreted.18,35 as a behavioral inhibitor that reduced attraction, rather than acti- The recent development of genome editing techniques in vating avoidance behavior itself.14,22 Additional electrophysiolog- Aedes aegypti allowed for the genetic analysis of the OR pathway ical and behavioral studies supported the inhibitory effect of in a mosquito.35 Aedes orco mutants did not respond to host odor DEET.23,24 Whether DEET can directly inhibit the as yet alone, but were still able to host-seek in the presence of carbon unidentified lactic acid receptor has not been shown. dioxide, demonstrating that redundant mechanisms exist for

46 Fly Volume 9 Issue 1 mosquitoes to sense hosts.35 Redundant mechanisms for mosquito host-seeking were also revealed by genetically ablat- ing carbon dioxide detection and testing responses of the mutants to diverse attractive stimuli.20 As in Drosophila, orco mutants were unaffected by the presence of DEET in the vapor phase, responding both to human odor and human skin treated with 10% DEET.35 Given that orco mutants can host-seek, if DEET simply masks host odors by blocking OR activation, it would not be an effective repellent. Therefore, current evidence argues that repellency by DEET does not involve the global inhibition of olfactory receptors.

The confusant hypothesis If DEET does not mask host odor detection by ORs, then another expla- nation is required. In early electrophysiological studies of Aedes aegypti mosquitoes, DEET increased the firing rate of olfactory receptor neurons in tri- choid short and long A2 sensilla, but inhibited the spontaneous activity of 40 medium-length sensilla. Neurons in basiconic A3 sensilla either did not show any response,14 or DEET inhibited neural spontaneous activity at low concentration, followed by excitation at a higher concentration.40 In coeloconic A4 sensilla, DEET inhibited neural spontaneous activity.40 These studies were based on the morphological and not the molecular identification of sensilla, (i.e., olfactory receptor neurons).41 Later, molecular studies revealed that with some exceptions, neurons in tri- choid and basiconic sensilla express Figure 1. Proposed hypotheses for how insect behavior is modulated by DEET in the vapor phase. (A) Human odor (yellow) binds to specific olfactory receptors (blue and light blue, but not red), activat- ORs, whereas neurons in coeloconic ing olfactory receptor neurons (ORNs) colored in green, which in turn activates glomeruli in the anten- 42 sensilla express IRs. Thus, DEET nal lobe (bright yellow) leading to host attraction. (B) DEET inhibits the sensation of host odor by likely activated, inhibited or had no binding to olfactory receptors and blocking the activation of ORNs (gray). (C) DEET modulates olfactory effect on OR-expressing neurons and receptor activation by human odor (blue and light blue, but not red), leading to changes in olfactory inhibited IR-expressing neurons, receptor neuron activation (gray and purple) that scramble odor coding by changing the normal activation pattern of glomeruli, and host attraction is blocked. (D) DEET binds to a specific olfactory recep- underscoring the complex role of tor (red) that is expressed in an ORN that activates a neural circuit that causes aversion. The aversive DEET in olfactory modulation. Yet signal overrides the neural activation pattern elicited by attractive cues sensed by other ORNs. Acti- again, the molecular players involved in vated olfactory receptor neurons are green, inactivated are gray, and modulated are purple. Odor these processes could only be inferred, plumes from the human host are indicated in shades of yellow. The molecule depicted is DEET. but not specifically identified. The promiscuous effects of DEET on olfaction have been supported by molecularly defined electro- depending on the olfactory receptor tested. Application of DEET physiological surveys of OR-expressing neurons15,18 and heterolo- itself had little to no effect on OR-expressing neurons gously expressed ORs.16,43-45 In these studies, DEET in some studies,15,18 but others have shown responses of ORs administered with odors could activate, inhibit, or have no effect, to DEET without odors.16,40,46,47 As insects are unlikely to

www.tandfonline.com Fly 47 experience DEET in the absence of host odors, the variation in its attractive stimulus, (ii) the inability of published behavioral assays effect across different ORs suggested that DEET disrupts olfactory to control for physical contact with DEET and (iii) the possibility coding by confusing the normal activation pattern elicited by host of insect specific differences, and hence the intrinsic difficulty for odor, and hence the confusant hypothesis was born18;Itposited direct comparison between studies using different insect species. that the sensitivity of multiple ORs to attractive odors was altered In addition, these hypotheses posit very different numbers of ORs by DEET, but DEET had little to no behaviorally relevant effect necessary for DEET repellency. The confusant hypothesis suggests on its own. In this model, if any inherent repellent activity of that many ORs are modulated by DEET. The smell and avoid DEET exists, that activity needs to be enhanced by host odor. The hypothesis suggests that only one OR may be activated by DEET. confusant hypothesis recognizes that DEET can inhibit the activa- This controversy is not easy to resolve. The genetic basis of DEET tion of some neurons, but suggests that the overall effect is not detection was discovered using orco mutants, which ablate the masking the host. Rather, host kairomones are still sensed but their function of all ORs at once. Conclusively determining the number interpretation by the insect is dramatically altered. of ORs required for DEET repellency would involve generating many new OR mutants. If multiple ORs are involved, insects that The smell and avoid hypothesis contain several OR mutations at once must be tested. Such hercu- Contradicting the findings showing that DEET had no effect lean efforts may be worthwhile, as identifying the odor-selective on its own or was attractive in several studies,15,22,48-50 acompet- ORs required for DEET repellency would provide new molecular ing mechanism for DEET action has been suggested. The smell targets for repellent design. and avoid hypothesis proposes that DEET is perceived as a nox- 51 ious odor by the insect. Labeled-line repellency of this kind has A role for IRs in DEET-driven repellency? been clearly demonstrated for the microbial odorant geosmin in A recent study suggests that IRs may also be necessary for 52 Drosophila. Similar to geosmin, DEET may be sensed by an DEET repellency.54 Kain et al.54 showed that DEET activates olfactory receptor that activates a neural circuit that elicits avoid- Ir40a-expressing neurons in the Drosophila sacculus, a ance behavior. Two studies in Culex quinquefasciatus mosquitoes 3-chambered pit beneath the antenna’s surface. In addition, 51,53 support this view. In the absence of odor cues, mosquitoes RNAi knockdown of Ir40a allowed flies to enter a 50% DEET- avoid a sugar solution they would normally feed on when it was perfumed trap that mock-treated flies avoid. Kain et al. also used 2 surrounded by a paper cylinder containing 1mg/cm of DEET. In chemical informatics to identify compounds that flies and mos- a similar assay mosquitoes also avoided an attractive heat source quitoes avoided. These compounds activated Ir40a neurons. 51 surrounded by a 10% DEET-treated paper ring. Both of these Tetanus toxin silencing of Ir40a neurons allowed flies to enter a behavioral assays did not prevent mosquitoes from contacting trap perfumed by these compounds that control flies avoid. DEET; thus, it is difficult to discern whether the repellency Taken together, these findings suggest that Ir40a can activate an observed was mediated by olfactory or gustatory cues. Recent elec- aversive neural circuit in insects. The higher concentration of trophysiology studies with a Culex OR, CquiOR136, showed that DEET used in these behavioral assays makes it difficult to com- it could be activated by DEET, as well as other insect repellents, pare them to behavioral studies examining the role of ORs in 53 such as PMD, Picaridin, and IR3535. CquiOR136 was also DEET repellency.15,35 It would be interesting to test if Orco is responsive to methyl jasmonate, a naturally produced insect repel- still necessary for repellency when concentrations of DEET are lent. This opens the possibility that the CquiOR136 pathway increased from 10% to 50%. Furthermore, studies in Culex mos- evolved to respond to repellents. Reducing the function of quitoes showed that knocking down Ir40a function using RNAi CquiOR136 via RNAi injection allowed Culex mosquitoes to be did not reduce DEET repellency.53 If Ir40a is required for the attracted to a heated blood-feeder surrounded by a paper soaked behavioral response to DEET, one must contemplate that both 53 with 0.1% DEET that mock injected mosquitoes avoided. This Ir40a and Orco are necessary for DEET sensation, but that nei- intriguing result suggests that only a single OR is the behaviorally ther pathway is sufficient for repellency on its own. This opens relevant DEET sensor in the vapor phase and that host odor may the possibility that Ir40a is a lower affinity DEET receptor that not be required for DEET to activate this receptor. However, this responds to high concentrations of DEET at close range. Addi- assay does not exclude the possibility of contact chemorepellency. tional loss-of-function studies in mosquitoes and other insects How volatile DEET interacts with ORs to alter olfactory sensi- will be necessary to determine the role of IRs in repellency. tivity is not yet clear. What is clear, however, is that DEET does not simply inhibit the detection of host odors by ORs. Instead, DEET may be a confusant that alters OR sensitivity to host odors Gustatory and contact modes of action or a labeled-line repellent that activates an OR specific for noxious odors. Discriminating between these 2 competing hypotheses will Most studies of DEET have focused on vapor repellency be useful for designing the next generation of repellents and for against flying insects. Tactile repellency has been studied mostly expanding our understanding of how repulsive behavior is gener- in crawling arthropods such as ticks.55 DEET has been shown to ated in insects. To do this, the role that host odors play in DEET be both an anti-feedant and a repellant on contact in insects. repellency must be clearly shown. Previous studies have failed to Whether the same molecular targets mediate these behaviors adequately address this question for a number of reasons, includ- remains unclear. What is clear is that DEET can alter behavior ing (i) the difficulty of quantifying behavior in the absence of an by multiple chemosensory modalities.

48 Fly Volume 9 Issue 1 DEET is an anti-feedant DEET-treated skin triggers a bitter taste response through GRs Humans perceive DEET as bitter.9 In Drosophila, GRs that or contact disengagement involving a distinct molecular pathway. sense bitter chemicals are necessary to avoid ingestion of DEET Identification of the contact receptor(s) would provide additional containing food.56 This avoidance occurs even with 0.1% DEET, molecular target(s) for chemical screens to identify new topical a significantly lower concentration than the chemical has been repellents that could block mosquito blood-feeding. used for olfactory-based laboratory studies or in commercial products. Similarly, other studies have shown that the mosquitoes do not feed on blood that contains a very small amount of DEET (0.065%), even though some of them penetrated their proboscis Implications for the Next Generation into a membrane feeder.57,58 As mosquitoes are unlikely to smell of Insect Repellents DEET in these assays, these results suggest that repellency occurs through labellar taste receptors, and not through an olfactory DEET is safe and effective, but has several drawbacks.4 It has mechanism. Repellency was also observed when DEET was to be applied at relatively high concentrations (10% or more) to applied to the feeding membrane, suggesting a role for taste neu- be effective. DEET needs to be reapplied to skin every few hours rons located on both the legs and the proboscis.58 to ensure repellency. Pure DEET melts plastic and vinyl. It is Evidence for gustatory receptors specifically detecting DEET also not very volatile. Because of the short-range spatial protec- was obtained from studies in Drosophila.56 It was found that aver- tion of DEET,50 it needs to be applied either on skin or cloths to sive taste neurons tuned to numerous bitter compounds also effectively repel arthropods, whereas other application methods, respond to DEET and that DEET-mediated activation of these such as wearing a DEET-impregnated wristband, do not work.4 neurons required 3 bitter taste receptors, including Gr66a. More- There is also evidence that mosquitoes can become resistant to over, behavioral experiments showed that the proboscis extension DEET.62 The limitations of DEET have fueled a search for reflex response induced by sugar solutions applied to tarsi of flies alternatives. is severely reduced when DEET was added to the sugar solution. The next generation of insect repellents are likely to be ratio- These observations indicate that DEET has an inhibitory effect nally discovered using molecular targets that enable insect host on a feeding response by activating bitter taste neurons that coun- attraction such as olfactory receptors. This approach allows for teract the activity of sweet sensing neurons. Electrophysiology high throughput screening of hundreds of thousands of com- recordings in Aedes aegypti have also shown that DEET activates pounds, many more than were screened to identify DEET.5,6 bitter taste neurons in the labellum that respond to many other ORs can be functionally expressed in cultured cells and their ion bitter compounds.59 Interestingly, many labellar gustatory recep- channel activity can be visualized.63,64 This allows for screening tor neurons also express AaegGR14, the putative ortholog of the of compounds that can directly activate ORs or change their sen- Drosophila bitter receptor GR66a.60 Whether the tarsi of Aedes sitivity to odor-ligands. Recent screens have already identified an respond to DEET remains to be seen, as AaegGR14, is not agonist of insect ORs, VUAA1.63,65 This chemical activates all expressed in neurons of sensilla located in tarsi. However, they OR-Orco complexes tested and likely works by directly interact- do express many GRs related to Drosophila bitter taste receptors ing with Orco. VUAA1’s main drawback is that it is not very vol- and hence are likely to respond to bitter compounds.60 Regard- atile. Continued effort will likely yield other compounds that can less, the current evidence strongly suggests that insects taste manipulate insect olfactory receptors and are volatile enough to DEET and avoid ingesting it like other bitter compounds. become candidate insect repellents. Identified compounds should posses a number of qualities to overtake the current gold stan- Human skin treated with DEET repels mosquitoes dard, DEET.3 These include: efficacy in the vapor phase at low The mechanism of DEET repulsion of mosquitoes when they concentrations, specificity for the receptor it was designed to tar- land on skin differs from DEET repellency in the vapor phase. get, low cost, easy to impregnate into wearable items, such as Aedes orco mutants are attracted to DEET-treated skin, but do wristbands, be long lasting, have a pleasant odor, and of course not blood feed.35 This result could be explained by 2 mecha- be non-toxic to humans and the environment. nisms: 1) that orco mutant mosquitoes need physical contact with Although the mechanism is not yet clear, DEET likely alters DEET-treated skin in order to be repulsed or 2) that there are the activity of olfactory receptors either in the context of odors or low-affinity olfactory receptors that sense DEET when the mos- on its own. This has several implications for any screening proto- quito is in close proximity to skin. To test this, video recordings col. Chemical screens designed to isolate candidate repellents will of orco mutant mosquitoes and wild-type controls documented need to seek chemicals that broadly inhibit multiple classes of host-seeking behavior at close-range. When DEET was applied insect odorant receptors due to the redundancy that exists in to skin, orco mutants landed on the skin and then left without insect olfaction.20,35 As an alternative, screens can seek to modu- biting. This result demonstrates that contact mediates the repul- late specific classes of olfactory receptors such as ORs that have sion in the absence of an intact olfactory system. Thus the orco been associated with repellency. Understanding which ORs are mutant allows the separation of the contact and olfactory mecha- modulated by DEET, and whether the changes are behaviorally nisms of DEET in the mosquito, with the potential caveat that relevant would do much to narrow the field of molecular targets other olfactory receptors, including IRs, are intact in this to be screened. In other words, to find volatile chemicals that mutant.35 It remains to be determined whether landing on trigger repellency, it is necessary to connect insect olfactory

www.tandfonline.com Fly 49 receptors with the behaviors they enable. Understanding how Fernando Noriega, Daria Siekhaus, Fredis Mappin, Emily DEET works may lead us to these important molecular targets. Dennis, Patrick Burke, Michael Perez, Reinier Alvarez, Christian Larsen, and Maria Areiza for their helpful comments on the man- uscript. I apologize in advance to all researchers whose work was Disclosure of Potential Conﬂicts of Interest not cited in this review. No potential conﬂicts of interest were disclosed.

Acknowledgments Funding I would like to thank Babak Ebrahimi for his assistance in Florida International University and a NIH/NIAID career developing this review. I would also like to acknowledge award (K22AI112585) support my work.

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